Flavor improvement for foodstuffs comprising treating foodstuffs with a compound of the group of 5(4)-amino-4(5)-imidazolecarboxamide riboside-5&#39;-phosphoric acid and non-toxic salts thereof



United States Patent FLAVOR IMPROVEMENT FOR FOODSTUFFS COM- PRISING TREATING FOODSTUFFS WITH A COM- POUND OF THE GROUP OF (4)-AMINO-4(5)- IMIDAZOLECARBOXAMIDE RIBOSIDE-5- PHOS- PHORIC ACID AND NON-TOXIC SALTS THERE- OF Hsing T. Huang, Groton, and Gilbert M. Shull, Old Lyme, Conn., assignors to Chas. Pfizer & Co, Inc., New York, N.Y., a corporation of Delaware No Drawing. Original application Aug. 28, 1963, Ser- No. 305,223. Divided and this application Dec. 7, 1966, Ser. No. 616,141

12 Claims. (Cl. 99-440) This application is a division of the copendin-g application Ser. No. 305,223, filed Aug. 28, 1963, now abandoned.

The present invention relates to a method for improving the flavor of foodstuffs. More particularly, it relates to a method for improving the flavor of foodstuffs by the addition thereto of 5(4)-amino-4(5)-imidazolecarboxamide riboside-5-phosphoric acid or its non-toxic salts. It further relates to a biochemical process for the production of 5 (4)-amino-4(5 -irnidazolecarboxamide riboside-5'-phosphoric acid .by the phosphorylation of 5(4)-amino-4(5)- imidazolecarboxamide riboside.

The use of flavor enhancers, that is, flavoring agents or condiments to bring out the natural flavor of foods and to enhance the taste or flavor of foods is well-known. Flavor enhancers, or chemical condiments or seasoning agents, as they are often referred to, such as monosodium glutamate and sodium succinate have found wide spread use in the home, restaurants and food processing industry. More recently, a new class of flavor enhancer has received in creasing attention, at least one of which appears destined for wide spread acceptance. This new class of compounds comprises the naturally occurring 5'-nucleotides all of which contain the purine nucleus. According to the literature the more active compounds Within this class are 5- inosinic acid (inosinic acid), 5'-guanylic acid and 5- xanthilic acid. Other compounds reportedly active as flavor enhancers are 5'-cytidilic and 5'-uridylic acid which contain the pyrimidine portion but not the fused imidazole ring system of the purines.

In a study on the relationship between the structure of inosinic acid and its taste enhancing property, Kuninaka (Yakkayoku 11, 1229/32 (1960)) determined that hypoxanthine and inosine which are precursors of inosinic acid are inactive as flavor enhancers as are the 2- and 3'- inosinic acids. Phosphorylation at the 5'-position of the ri bose moiety is essential for flavor enhancement.

It has now been unexpectedly and surprisingly found that 5(4)-amin o-4(5)-imidazolecarboxamide riboside-5'- phosphoric acid and its non-toxic salts are potent flavor enhancers and are at least as effective as inosinic acid. They are of value as flavor enhancers (condiments or seasoning agents) for improving the taste or flavor of any form of foodstuffs, such as soups, sauces, pastes, canned foods, boiled foods, pickled foods, beverages, breads, juices, cereals, meat products, dairy products, pastries and sweets, but are more highly suited for certain types of foodstuffs such as soups, meats, sauces and pastes. In some instances these materials may also affect the aroma of foods. This appears to be particularly true in the case of soups and broths.

5(4)-amino 4(5) imidazolecarboxamide riboside-S phosphoric acid and its non-toxic salts are of great economic potential, particularly as regards the utilization of special diets and food sources which are presently not accepted 'by all people. The flavor of such food sources is generally responsible for their poor acceptance. 5(4)- amino-4(5)-imidazolecarboxamide riboside-5'-phosphoric acid and its non-toxic salts, by virtue of their ability to bring out, to blend and modify flavors, to enhance existing flavors and to suppress undesirable flavors, may overcome the objections to such foods and thus improve th utilization of available foodstuffs.

5 (4)-amino-4(5) imidazolecarboxamide riboside 5'- phosphoric acid forms a wide variety of salts with both inorganic and organic bases, including basic amino acids such histidine and arginine. 5(4)-amino-4(5)-imidazolecarboxamide riboside 5'-phosphoric acid itself, when placed as a small quantity upon the tongue, produces an immediate and long lasting sensation. The taste is primarily meaty and mouthfilling followed by a somewhat astringent sensation. The disodium salt of a 5 (4)-amino- 4(5)-imidazolecarboxarnide riboside-5'-phosphoric acid and in fact the non-toxic salts of 5(4)-amino-4(5)-imidazolecarboxamide riboside-5-phosphoric acid produce substantially the same taste sensation, per se indicating that the taste enhancing property is due to the 5(4)- amino-4(5 -imidazolecarboxamide riboside-5'-phosphoric acid anion itself. The alkali metal salts appear to bring about a more immediate effect than does the acid form probably because of their greater solubility, rate and de gree of ionization. Dilute aqueous solutions of 5(4)- amino-4 (5 -imidazolecarboxamide riboside-5'-phosphoric acid and its non-toxic salts are readily distinguishable tastewise from water itself.

The disodium salt of 5(4)-amino-4(5)-imidazolecarboxamide riboside-5'-phosphoric acid and the free acid are the preferred forms since they are easily and economically prepared and afford the same tactile sensations as do other salts. However, it is contemplated within the purview of this invention that other non-toxic salts such as the potassium, calcium, magnesium, aluminum, the ammonium, histidine, lysine and arginine salts are substantially equivalent to the disodium salt as regards their flavor enhancing property.

5(4)-amino-4(5) -i1nidazolecarboxamide riboside 5' phosphoric acid and its non-toxic salts may be used per se in a given foodstuff or may :be used as mixtures of tWO or more salts or in mixture with other flavor enhancers such as monosodium glutamate, sodium succinate and 5'- guanylic and inosinic acids and salts thereof. For example, disodium 5 (4)-amino 4(5) -imidazolecarboxamide riboside-5'-phosphoric acid may be used in combination with monosodium glutamate, sodium-5-guanylate or disodlum inosinate. In such instances the improvement in taste or flavor realized appears to "be due to co-action between the flavoring agents and is frequently greater than the effect of either enhancer per se.

For convenience of use, particularly by the individual user, disodium-5(4)-a-mino 4(5) imidazolecarboxamide riboside-S'phosphoric acid may be mixed with a suitable carrier or diluent such as sodium chloride, magnesium carbonate, spices, malt, calcium stearate, hickory smoked dried yeast and dextrose. The concentration of the ribotide in such compositions can, of course, vary widely: however, concentrations of from about 5% to about by weight of 5(4)-amino-4(5)imidazolecarboxamide 3 riboside-'-phosphoric acid have been found satisfactory. Higher or ilower concentration afford no advantages.

The quantity of disodium 5(4)-amino-4(5)-imidazolecarboxamide riboside-5'-phosphoric acid to be added to a given foodstuff depends primarily on the nature of the foodstuff and is easily determined by a simple taste test of the thus treated foodstuff. It has been noted that a several fold difference may exist between the amount of disodium 5(4)-amino-4(5)-amidazolecarboxamide riboside- 5-phosphoric acid sufficient to bring about a significant change in flavor of a given foodstuff and that which will effect maximum flavor improvement. However, since an excess of the flavor enhancer imparts no unpleasant taste to the foodstuff this problem is economic rather than organoleptic in nature. Similar remarks apply to 5(4)- amino4(5)imidazoleearboxamide riboside-5'-phosphoric acid and its other non-toxic salts since, as previously mentioned, the 5(4)-amino-4(5)-imidazolecarboxamide riboside-5'-phosphoric acid anion is the active moiety.

The flavor enhancing property of 5(4)-amin-o-4(5)- imidazolecarboxamide riboside-5-phosphoric acid and its non-toxic salts is detectable in most foodstuffs at concentrations of about 0.005%. In beverages, concentrations of, as low as 0.001%, may bring about noticeable improvements in flavor. In general, concentrations of from about 0.001% to about 0.1% are suitable for most foodstuffs.

5(4)-amino-4(5)-imidazolecarboxamide riboside 5- phosphoric acid can be produce-d by enzymatic procedures as is well known. Greenberg, (J. Biol. Chem., 219, 423) for example, phosphorylates 5(4)-amino-4(5)-imidazolecarboxamide riboside by a mixture of enzyme extracts prepared from brewers yeast and muscle using adenosine triphosphate as phosphorylating agent. Flaks et al. (J. Chem. Soc, 76, 2275) form the ribotide by reacting inosinic acid with glycine in the presence of an enzyme isolated from pigeon liver extract and in still another method by the direct condensation of 5(4)-amino-4(5)- imidazoleearboxamide and 5'-phosphoribosyl pyrophosphate, (J. Biol. Chem., 228, 201). More recently a chemical method for its preparation was reported by Shaw (J. Am. Chem. Soc., 83, 4770) utilizing inosine as starting material.

The known methods for the preparation of 5 (4)-amino- 4(5) imidazolecarboxamide riboside-5'-phosphoric acid including the enzymatic phosphorylation of 5(4)-amino- 4(5)-imidazolecarboxamide riboside require highly purified systems and are unsuitable for large scale commercial production.

It has now been found 5(4)-amino-4(5)-imidazolecarboxamide riboside can be conveniently and economically phosphorylated by incubation with an orthophosphate in the presence of an actively fermenting brewers yeast. Adenosine may be added to the fermentation in catalytic amounts, e.g., 1% of the substrate or less, since it frequently accelerates the phosphorylation. Under the conditions of the present process phosphorylation of the 5(4)- amino-4(5)-imidazolecarboxamide riboside is substantially complete. Ninety to ninety-five percent of the phosphorylation product is 5(4)amino-4(5)-imidazolecarboxamide riboside-5'-phosphoric acid. A small amount of more highly phosphorylated products, presumably diand triphosphorylated products, are also obtained. The process, which is readily adapted to large scale production, is more fully described in the copending application filed concurrently herewith, Serial No. 616,142.

The following examples are given by way of illustration and are not to be construed as limitations of this invention, many variations of which are possible without departing from the scope and spirit thereof.

PREPARATION A Brewers yeast, used in the brewing of lager beer and received from the brewery as a pressed cake, is grated through a -mesh screen, dri d i air o g t en stored in a refrigerator. grams of this dried yeast is added to two liter flask containing Glucose g 15.0 Disodium hydrogen phospsate g 12.8 Dihydrogen sodium phosphate g" 8.28 Tap water (30 C.) ml 900 and the flask placed in a constant temperature bath (30 C.) and magnetically stirred for 30 minutes. Then, the following materials are added:

Glucose g 10.0 Alcar 1 g 9.3 Adenosine' mg 50 Carbon tetrachloride mg 10 Toluene mg 10 1 5 (4) amino-4 5)-imidazolecarboxamlde riboside.

Papergrams are run every half hour by spotting a one microliter portion of the reaction mixture and developing in the system methyl ethyl -ketone (9):acetic acid (2.5 )1 water (3) for one half hour. The paper-gram is dried and sprayed with Bratton and Marshall reagent. R) of the riboside is 0.6 and of the ribotide is 0.25.

The system, pH 6.2, is stirred at 30 C. for 2 hours after which 5.0 g. glucose is added. (The voluminous foam produced gradually subsides.) The stirring is continued an additional 7 hours until reaction is complete, then the reaction (pl-I 6.0) refrigerated overnight. It is then centrifuged, decanted and the residue washed three times with Water (3X 300 ml.). Paper chromatography of the supernatant and each of the three wash liquors in the system, methyl ethyl ketone:acetic acidzwater (9: 2.5:3), followed by Bratton-Marshall test of the chromatograms, gives a positive test for the ribotide. A fourth water wash of the residue is found to contain only a trace of ribotide.

PREPARATION B The phosphorylation process of Preparation A is repeated in duplicate and the combined supernatants and wash liquors (4.1 liters) passed through a column of Dowex-l, formate cycle, column size 2 liters resin, prepared by regenerating Dowex-l, chloride cycle, with 2 liters of 2 N sodium formate followed by washing with 12 liters of distilled water. Eight hundred ml. fractions are taken at the rate of one every 20 minutes. The column is washed with 8 liters of distilled water at the above rate. Paper chromatography of the individual fractions shows only a trace of riboside and no ribotide to be present.

The column is then eluted with Z N formic acid at the rate of a 200 ml. fraction every 15 minutes until the eluate contains only a slight trace of the ribotide. The fractions containing the ribotide (determined by paper chromatography) are combined and freeze dried. The fractions (35-46) obtained after eluting with about 4 liters of formic acid produce, on standing in the refrigerator, crystalline ribotide as the monohydrate. The crystalline ribotide is filtered off, washed with methanol and dried (7.0 -g.). The filtrate is combined with the remaining eluate and freeze dried. The freeze dried residue is suspended in 200 ml. distilled water, stirred for 30 minutes and 50 ml. more distilled water added. The suspension is then cooled in an ice-water bath, stirred for 30 minutes and filtered. The cake is washed with methanol and air dried (14.6 g.). The aqueous filtrate can be freeze dried and re-worked as above.

Total yield=21.6 g. (86.7% based on the riboside used; 90.2% recovery from the broth).

Analysis.-Calcd. for C H N O RH O: C, 30.3%; H, 4.8%; N, 15.7%. Found: C, 30.0%; H, 5.1%; N, 15.5%.

Molecular Weight of potentiometric titration=362. Calculated value for the monohydrate is 356.2. The ultraviolet absorption spectra of both crops agrees with the literature values.

PREPARATION c A nutrient medium having the following composition is prepared and sterilized at 120 C. for 30 minutes.

Distilled water to volume.

Inoculum is prepared by transferring Saceharomyces cerevisiae ATCC 11795 to a 300 ml. Erlenmeyer flask containing 50 ml. of the above medium. The inoculated flask is incubated stationary at 28 C. for one day with gentle shaking by hand every four hours. The entire culture is then transferred to a Fernbach flask containing one liter of the same medium. Following the same stationary incubation process as before the yeast is used to inoculate a fermenter containing liters of medium. The same stationary technique with gentle shaking or stirring every four hours is used. The yeast cells are collected by centrifugation and washed with ice-cold water.

Ninety-four ml. of yeast cells prepared in this manner are slurried in 70 ml. of water and added to a flask containing 3.75 g. dextrose, 3.2 g. disodium hydrogen phosphate, 2.07 g. sodium dihydrogen phosphate monohydrate and 50 ml. water. The volume is made up to 250 ml. and the mixture stirred at 30 C. for 30 minutes. Then, 2.0 g. dextrose, 2.58 g. 5(4)-amino-4(5)-imidazolecarboxarnide riboside, 100 mg. adenosine, 2.5 ml. carbon tetrachloride and 2.5 ml. toluene are added, the mix ture stirred at 30 C. for 2 hours, and 1.75 g. dextrose added. The reaction is followed by means of paper chromatography as in Preparation A. The reaction mixture is centrifuged after 4.5 hours and the yeast cells washed with water. The combined wash and centrifugate is worked up by the procedure of Preparation B.

PREPARATION D To 0.1 mole of 5(4) amino-4(5)-imidazolecarboxamide ri'boside-5'-phosphoric acid in 25 ml. water there is added 0.02 mole of sodium hydroxide as a 1 N solution. The resulting solution is filtered if necessary then freeze dried to give the disodium salt.

In like manner, but substituting equimolar quantities of potassium hydroxide, ammonium hydroxide, magnesium hydroxide, calcium hydroxide, aluminum hydroxide, histidine, lysine and arginine for sodium hydroxide the corresponding dipotassium, diammonium, magnesium, calcium, aluminum, histidine, lysine, and arginine salts are produced.

PREPARATION E The procedure of Preparation D is repeated but using 0.01 mole of sodium hydroxide in place of 0.02 mole to give the monosodium salt.

In like manner, the monoammonium, monopotassium, monohistidine, monolysine and monoarginine salts are prepared using one molar proportion of the appropriate base.

Example I To one liter of beef bouillion there is added 100' mg. (0.01% by weight) of 5 (4)-arnino-4(5 )-imidazolecarboxamide riboside-5'-phosphoric acid and the mixture cooked to provide a bouillion having improved flavor and aroma.

Example II To a liter of chicken broth is added a suitable amount of table salt and spices commonly used to prepare soup and 200 mg. (0.02% by weight) of disodium-5 (4)-amino- 6 4(5)-imidazolecarboxamide riboside-5'-phosphoric acid. The finished soup is of considerably improved flavor.

Example III To one liter of the chicken broth referred to in Example II there is added suitable amounts of table salt and spices used to prepare soup. mg, of disodium 5 (4)- amino 4(5) imidazolecarboxamide riboside-5-phosphoric acid and 100 mg. of disodium inosinate to give a finished soup of much improved flavor.

Example IV 'Vegetable soup seasoned with a suitable quantity of table salt is further treated with 0.02% by weight of disodium 5 (4)-amino-4(5)-imidazolecarboxamide riboside- 5'-phosphoric acid and 0.05% by weight of monosodium glutamate. The mixture is cooked to provide a soup of better taste or flavor.

Example Beef bouillion is seasoned with a suitable quantity of table salt and further seasoned with 0.02% by weight of disodium 5(4)-amino-4(5)-imidazolecarboxamide riboside-5'-phosphoric and 0.01% by weight disodium-5- guanylate to give a boullion of improved flavor and aroma.

Example VI One liter quantities of the beef bouillon referred to in Example II are treated with one of the following salts of 5(4) amino-4(5)-imidazolecarboxamide riboside-5'- phosphoric acid or one of the combinations of flavor enhancers listed below. The quantities added are given in percent by weight.

In each instance improved flavor is imparted to the bouillon.

(0.015%) plus disodium-5'guanylate Example VII An 8-ounce slice of roast beef, rare, is seasoned with 0.1 g. of a mixture comprising equal quantities by weight of disodium 5 (4) amino 4(5)-imidazolecarboxamide riboside-5'-phosphoric acid and sodium chloride to provide meat of considerably improved flavor.

Example VIII An 8-ounce charcoal broiled hamburger patty is seasoned with 0.1 g. of a mixture comprising 10% disodium 5 (4) amino 4 (5) imidazolecarboxamide riboside-5- phosphoric acid, 70% monosodium glutamate, 18% table salt, and 2% calcium stearate to provide a hamburger patty having improved flavor.

Example IX An 8-ounce hamburger patty, charcoal broiled, is seasoned with a mixture comprising 50% disodium 5(4)- amino-4 5 -imidazolecarboxamide riboside-S '-phosphoric acid, 40% table salt, 8% hickory smoked dried yeast and 2% calcium stearate to provide a hamburger having improved taste.

Example X In a test designed to determine the flavor enhancing power of disodium 5(4)-amino-4(5)-imidazolecarboxamide riboside--phosplioric acid (disodium AlCARP) relative to that of disodium inosinate, a triangular taste test is arranged. Three samples of each of the several finished soups listed below'are taken. To one sample of each of the soups there is added disodium 5(4)-amino-4(5)- imidazolecarboxamide riboside-5'-phosphoric acid on a. percent by weight basis in the amounts indicated. To a second sample of each of thelsoups equal amounts by Weight of disodium inosinate are added. The third sample of each soup is not treated and is the odd sample.

Sample 1 Sample 2 Sou Disodium Disodium AlCARI, Percent Inosinate, Percent Chicken noodle 0.03 0.03 Beef bouillon 0. 028 0. 028 Onion l 0.0 0.02

0. 015 0. 015 Cream of mushroom 0.032 0.032

A mixture comprising equal parts by weight of disodium 5 (4)-amino-4(5 -imidazolecarboxamide ribo'side-5-phosphoric acid and disodium-5-guanylate is added to each of several finished soups on a weight basis as indicated.

Soup: Mixture (percent by weight) Chicken noodle 0.0075 Cream of mushroom 0.032 Tomato 0.015 Onion 0.02 Beef bouillon 0.005 Onion 0.01 Tomato 0.005 Cream of mushroom 0.008

In each instance, a soup of improved flavor i produced.

Example XII A composition comprising by weight of disodium 5(4) -amino-4(5 )-imidazolecarboxamide riboside-5-phosphoric acid dihydrate and 90% by weight of monosodium glutamate is used to season sliced roast beef. The resulting meat is of much improved flavor. The flavor enhancing effect is obtained on both hot and cold roast beef.

Example XIII Sliced roast beef is seasoned with a composition comprising 95% monosodium glutamate, 5% disodium 5(4)- amino-4(5 -imidazolecarboxamide riboside-5-phosphoric acid and 5% disodium 5'-guanylate to provide meat of markedly improved flavor. The composition is effective on 'both hot and cold beef.

What is claimed is:

1. A 'processtor improving the flavor of a foodstuff which comprises treating'said foodstuff with a compound selected from the group Consisting of 5(4)-amino-4(5- imidazolecarboxamide riboside-5-phosphoric acid and the non-toxic salts thereof.

2. A process as claimed in claim 1 wherein disodium 5 (4)-amino-4 5 -imidazolecarboXamide riboside-5-phosphoric acid is added to the foodstuff.

3. A process as claimed in claim 1 wherein 5 (4)-amino- 4(5)-imidazolecarboxamide riboside-5'-phosphoric acid is added to the foodstuff.

4. A process as claimed in claim 1 wherein disodium inosinate is also added to the foodstuff.

5. A process as claimed in claim 1 wherein monosodium glutamate is also added to the foodstuff.

6. A process a claimed in claim 1 wherein disodium- 5'-guany1ate is also added to the foodstuff.

7. A composition for improving the flavor of foodstuffs which comprises disodium inosinate and a compound selected from the group consisting of 5(4)-amino-4(5)- imidazolecarboxamide riboside-5'-phosphoric acid and the non-toxic salts thereof together with a carrier.

8. A composition for improving the flavor of foodstufls which comprises monosodium glutamate and a compound selected from the group consisting of 5(4)-amino- 4(5)-imidazolecarboxamide riboside-5-phosphoric acid and the non-toxic salts thereof together with a carrier.

9. A composition for improving the flavor of foodstuffs which comprises disodium-5'-guanylate and a compound selected 'from the group consisting of 5(4)-amino-4(5)- imidazolecarboxamide riboside-5-phosphoric acid and the non-toxic salts thereof together with a carrier.

10. A composition for improving the flavor of foodstuffs which comprises disodium-5-guanylate, monosodium glutamate and a compound selected from the group consisting of 5(4)-amino-4(5)-imidazolecarboxamide riboside-S' phosphoric acid and the non-toxic salts thereof together with a carrier.

11. Foodstuffs seasoned with a compound elected from the group consisting of 5(4)-amino-4(5)-imidazolecarboxamide riboside-5'-phosphoric acid and its non-toxic salts.

12. Foodstuffs seasoned with disodium 5(4)-amino- 4(5 )-imidazolecarboxamide riboside-5'-ph0sphoric acid.

References Cited UNITED STATES PATENTS A. LOUIS, MONACELL, Primary Examiner.

H. H. KLARE, Assistant Examiner. 

1. A PROCESS FOR IMPROVING THE FLAVOR OF A FOODSTUFF WHICH COMPRISES TREATING SAID FOODSTUFF WITH A COMPOUND SELECTED FROM THE GROUP CONSISTING OF 5(4)-AMINO-4(5IMIDAZOLECARBOXAMIDE,RIBOSIDE-5''-PHOSPHORIC ACID AND THE NON-TOXIC SALTS THEREOF. 